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1.
Mol Biol (Mosk) ; 54(2): 300-307, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392200

RESUMO

The thermal stability of protein enzymes is determined in vitro by measuring the enzymatic activity during incubation at constant temperature. Refolding of thermal inactivated enzymes is carried out both in vitro and in vivo, in the presence of chaperones, usually at temperature optimal for the particular enzyme for the manifestation of enzymatic activity. In the present work thermal stability of enzymes in vitro (using purified preparations) and in vivo (directly in the bacterial cell) has been determined. Bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi and Photorhabdus luminescens as protein substrates have been used. It is shown that the thermal stability of the P. luminescens and P. leiognathi luciferases in vivo in the Escherichia coli MG1655 dnaK^(+) and PK202 ΔdnaKJ14 strains is considerable higher than the thermal stability of "cell-free extract" luciferases. When an uncoupler of oxidative phosphorylation the carbonyl-cyanide-3-chlorophenylhydrazone (CCCP) that reduce the intracellular concentration of ATP to a minimum level, and the volatile hydrophobic substance (-)-Limonene (C10H16) as an inhibitor of chaperone-dependent refolding are added to the medium, the thermal stability of luciferases reduces almost to the level which is characteristic for the purified protein preparation. It is shown that the ATP-dependent chaperones ClpA and ClpB are essential for the increase of thermostability of luciferases in bacterial cells. Also, it is shown that the DnaKJE-dependent refolding of thermoinactivated luciferases is practically absent if the protonophore СССР or the hydrophobic substance (-)-Limonene was added to the bacterial suspension. Taking the data presented in this paper into account, it is necessary to consider the presence in bacterial cells of two different groups of ATP-dependent chaperones: 1st group (DnaKJE, GroEL/ES) is able to conduct the refolding both at low temperature after protein thermal inactivation and at high temperature at which protein thermal inactivation occurs; 2nd group (ClpA,ClpB, and possibly still unknown chaperones) is unable to conduct the standard refolding (i.e. at low temperature), but capable due to the hydrolysis energy of ATP of maintaining nonequilibrium stabilization of protein native forms at high temperature.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Chaperonas Moleculares/química , Dobramento de Proteína , Endopeptidase Clp , Estabilidade Proteica , Temperatura
2.
Adv Exp Med Biol ; 1194: 351-358, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468551

RESUMO

Protein homeostasis is a dynamic network that plays a pivotal role in systems' maintenance within a cell. This quality control system involves a number of mechanisms regarding the process of protein folding. Chaperones play a critical role in the folding, refolding, and unfolding of proteins. Aggregation of misfolded proteins is a common characteristic of neurodegenerative diseases. Chaperones act in a variety of pathways in this critical interplay between protein homeostasis network and misfolded protein's load. Moreover, ER stress-induced cell death mechanisms (such as the unfolded protein response) are activated as a response. Therefore, there is a critical balance in the accumulation of misfolded proteins and ER stress response mechanisms which can lead to cell death. Our focus is in understanding the different mechanisms that govern ER stress signaling in health and disease in order to represent the regulation of protein homeostasis and balance of protein synthesis and degradation in the ER. Our proposed model describes, using hybrid modeling, the function of chaperones' machinery for protein folding.


Assuntos
Modelos Biológicos , Chaperonas Moleculares , Dobramento de Proteína , Humanos , Chaperonas Moleculares/química , Doenças Neurodegenerativas/fisiopatologia , Biossíntese de Proteínas , Proteínas/metabolismo , Deficiências na Proteostase , Transdução de Sinais , Resposta a Proteínas não Dobradas
3.
Nat Struct Mol Biol ; 27(4): 363-372, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231288

RESUMO

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.


Assuntos
Proteínas de Choque Térmico HSP27/química , Chaperonas Moleculares/química , Proteína FUS de Ligação a RNA/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/isolamento & purificação , Humanos , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Fosforilação , Ligação Proteica/genética , Domínios Proteicos/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/genética
4.
Nat Commun ; 11(1): 1909, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312993

RESUMO

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.


Assuntos
Proteínas de Transporte/química , Antígenos de Histocompatibilidade Classe I/química , Proteínas de Membrana Transportadoras/química , Chaperonas Moleculares/química , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Alelos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Biblioteca Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Celular , Imunoquímica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Linfócitos T
5.
Nat Commun ; 11(1): 1504, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198371

RESUMO

The conserved ribosome-associated complex (RAC) consisting of Zuo1 (Hsp40) and Ssz1 (non-canonical Hsp70) acts together with the ribosome-bound Hsp70 chaperone Ssb in de novo protein folding at the ribosomal tunnel exit. Current models suggest that the function of Ssz1 is confined to the support of Zuo1, however, it is not known whether RAC by itself serves as a chaperone for nascent chains. Here we show that, via its rudimentary substrate binding domain (SBD), Ssz1 directly binds to emerging nascent chains prior to Ssb. Structural and biochemical analyses identify a conserved LP-motif at the Zuo1 N-terminus forming a polyproline-II helix, which binds to the Ssz1-SBD as a pseudo-substrate. The LP-motif competes with nascent chain binding to the Ssz1-SBD and modulates nascent chain transfer. The combined data indicate that Ssz1 is an active chaperone optimized for transient, low-affinity substrate binding, which ensures the flux of nascent chains through RAC/Ssb.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/química , Modelos Moleculares , Chaperonas Moleculares/química , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
6.
Proc Natl Acad Sci U S A ; 117(14): 7814-7823, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198203

RESUMO

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an "ADP-like" undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70-Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Conformação Proteica , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Transferência Ressonante de Energia de Fluorescência , Heterogeneidade Genética , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/química , Humanos , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Ligação Proteica/genética , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica/genética
7.
Biochemistry (Mosc) ; 85(1): 80-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079519

RESUMO

Here, we determined qualitative and quantitative characteristics of the chaperone and immunoglobulin-binding activities of recombinant Skp protein (rSkp) from Yersinia pseudotuberculosis using the methods of dynamic light scattering and surface plasmon resonance. Commercial human polyclonal IgG and Fc and Fab fragments of human IgG were used as substrate proteins. The activity of rSkp strongly depended on the medium pH. The most stable low-molecular-weight complexes with a hydrodynamic radius up to 10 nm were formed by rSkp and protein substrates at acidic pH values. Under these conditions, rSkp exhibited the lowest propensity to self-association and the highest affinity for human IgG and its Fc and Fab fragments, as well as prevented their aggregation most efficiently (i.e., demonstrated the maximal chaperone activity). As the medium pH increased, the affinity of rSkp for IgG and its fragments decreased; rSkp was not able to completely prevent the aggregation of protein substrates, but significantly slowed it down. The obtained information may be of practical interest, since the stability of therapeutic IgG preparations affects their safety and efficacy in medical applications.


Assuntos
Proteínas de Bactérias/química , Chaperonas Moleculares/química , Proteínas Recombinantes/química , Proteínas Quinases Associadas a Fase S/química , Yersinia pseudotuberculosis/metabolismo , Clonagem Molecular , Escherichia coli/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Dobramento de Proteína
8.
Nat Struct Mol Biol ; 27(2): 202-209, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042153

RESUMO

The mitochondrial membrane-bound AAA protein Bcs1 translocate substrates across the mitochondrial inner membrane without previous unfolding. One substrate of Bcs1 is the iron-sulfur protein (ISP), a subunit of the respiratory Complex III. How Bcs1 translocates ISP across the membrane is unknown. Here we report structures of mouse Bcs1 in two different conformations, representing three nucleotide states. The apo and ADP-bound structures reveal a homo-heptamer and show a large putative substrate-binding cavity accessible to the matrix space. ATP binding drives a contraction of the cavity by concerted motion of the ATPase domains, which could push substrate across the membrane. Our findings shed light on the potential mechanism of translocating folded proteins across a membrane, offer insights into the assembly process of Complex III and allow mapping of human disease-associated mutations onto the Bcs1 structure.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Chaperonas Moleculares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico
9.
Proc Natl Acad Sci U S A ; 117(2): 1036-1041, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888993

RESUMO

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.


Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Exocitose/fisiologia , Cinética , Fusão de Membrana/fisiologia , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
10.
Biochem J ; 477(2): 477-489, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31904842

RESUMO

Protein quality control is crucial for maintaining cellular homeostasis and its dysfunction is closely linked to human diseases. The post-translational protein quality control machinery mainly composed of BCL-2-associated athanogene 6 (BAG6) is responsible for triage of mislocalized membrane proteins (MLPs). However, it is unknown how the BAG6-mediated degradation of MLPs is regulated. We report here that PAQR9, a member of the Progesterone and AdipoQ receptor (PAQR) family, is able to modulate BAG6-mediated triage of MLPs. Analysis with mass spectrometry identified that BAG6 is one of the major proteins interacting with PAQR9 and such interaction is confirmed by co-immunoprecipitation and co-localization assays. The protein degradation rate of representative MLPs is accelerated by PAQR9 knockdown. Consistently, the polyubiquitination of MLPs is enhanced by PAQR9 knockdown. PAQR9 binds to the DUF3538 domain within the proline-rich stretch of BAG6. PAQR9 reduces the binding of MLPs to BAG6 in a DUF3538 domain-dependent manner. Taken together, our results indicate that PAQR9 plays a role in the regulation of protein quality control of MLPs via affecting the interaction of BAG6 with membrane proteins.


Assuntos
Homeostase/genética , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Receptores de Progesterona/genética , Humanos , Proteínas de Membrana/química , Chaperonas Moleculares/química , Ligação Proteica/genética , Domínios Proteicos/genética , Transporte Proteico/genética , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Progesterona/química , Ubiquitinação/genética , Ubiquitinas/química , Ubiquitinas/genética
11.
Nat Struct Mol Biol ; 27(2): 142-149, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31988523

RESUMO

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe-2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Chaperonas Moleculares/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química
12.
Nature ; 578(7794): 317-320, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996849

RESUMO

The ability to reverse protein aggregation is vital to cells1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp704,5. However, the model is also controversial, as the necessary motor activity has not been identified6-8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore11,12. Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)13 and degradation by the 26S proteasome14.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Agregados Proteicos , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Fluorescência , Proteínas de Choque Térmico/química , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Pinças Ópticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Redobramento de Proteína , Ubiquitina/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(4): 2099-2107, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31953264

RESUMO

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Chaperonas Moleculares/química , Vírus da Raiva/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Regulação Viral da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Transcrição Genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
14.
Biochem J ; 477(3): 629-643, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31939601

RESUMO

Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility-mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone ß-casein (ß-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between ß-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility-mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of ß-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein-chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms.


Assuntos
Caseínas , Lactalbumina , Chaperonas Moleculares , Agregados Proteicos/fisiologia , Amiloide/metabolismo , Caseínas/química , Caseínas/metabolismo , Lactalbumina/antagonistas & inibidores , Lactalbumina/química , Lactalbumina/metabolismo , Espectrometria de Massas , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteostase/fisiologia
15.
Proc Natl Acad Sci U S A ; 117(6): 2923-2929, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31974309

RESUMO

Small heat shock proteins (sHSPs) are a class of ATP-independent molecular chaperones that play vital roles in maintaining protein solubility and preventing aberrant protein aggregation. They form highly dynamic, polydisperse oligomeric ensembles and contain long intrinsically disordered regions. Experimental challenges posed by these properties have greatly impeded our understanding of sHSP structure and mechanism of action. Here we characterize interactions between the human sHSP HspB1 (Hsp27) and microtubule-associated protein tau, which is implicated in multiple dementias, including Alzheimer's disease. We show that tau binds both to a well-known binding groove within the structured alpha-crystallin domain (ACD) and to sites within the enigmatic, disordered N-terminal region (NTR) of HspB1. However, only interactions involving the NTR lead to productive chaperone activity, whereas ACD binding is uncorrelated with chaperone function. The tau-binding groove in the ACD also binds short hydrophobic regions within HspB1 itself, and HspB1 mutations that disrupt these intrinsic ACD-NTR interactions greatly enhance chaperone activity toward tau. This leads to a mechanism in which the release of the disordered NTR from a binding groove on the ACD enhances chaperone activity toward tau. The study advances understanding of the mechanisms by which sHSPs achieve their chaperone activity against amyloid-forming clients and how cells defend against pathological tau aggregation. Furthermore, the resulting mechanistic model points to ways in which sHSP chaperone activity may be increased, either by native factors within the cell or by therapeutic intervention.


Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas tau/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Ligação Proteica , Domínios Proteicos , alfa-Cristalinas/metabolismo , Proteínas tau/genética
16.
Nucleic Acids Res ; 48(3): 1531-1550, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31807785

RESUMO

FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.


Assuntos
Proteínas de Arabidopsis/ultraestrutura , Histonas/genética , Chaperonas Moleculares/ultraestrutura , Nucleoplasminas/química , Proteínas de Ligação a Tacrolimo/ultraestrutura , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Montagem e Desmontagem da Cromatina/genética , Cristalografia por Raios X , Histonas/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Nucleoplasminas/genética , Nucleossomos/química , Nucleossomos/genética , Peptidilprolil Isomerase/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Dobramento de Proteína , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética
17.
J Clin Invest ; 130(2): 699-714, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31845908

RESUMO

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.


Assuntos
Proteínas de Choque Térmico , Ivermectina , Chaperonas Moleculares , Neoplasias Experimentais , Receptor ErbB-2 , Células A549 , Animais , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ivermectina/química , Ivermectina/farmacologia , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Domínios Proteicos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
18.
J Biotechnol ; 307: 131-138, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31705934

RESUMO

The soluble expression of recombinant proteins in Escherichia coli is vital for protein applications in biotechnology and pharmaceuticals. However, the use of E. coli for efficient heterologous protein expression is hampered by several factors, such as poor expression and protein aggregation. Changing the culture or purification conditions may alleviate these issues, but methods based on gene fusion technology offer unique opportunities to improve the production and purification of soluble proteins. Here, we develop a novel fusion tag based on Spy, a newly identified molecular chaperone that functions in the periplasm of E. coli in an ATP-independent manner to prevent protein aggregation and assist in protein folding. We found that the tandem fusion of Spy stands among the well-described best fusion partners, such as MBP and SUMO, in increasing the soluble steady-state levels of six heterologous passenger proteins. Moreover, an easily aggregated passenger protein remained soluble after the removal of the Spy tag, implying that chaperone-dependent folding occurred when the passenger protein was fused to Spy. Our work expands the toolkit of fusion tags and allows them to aid in the production of unstable proteins with industrial or clinical values.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Formiato Desidrogenases/metabolismo , Proteínas Periplásmicas/metabolismo , Biotecnologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Formiato Desidrogenases/química , Formiato Desidrogenases/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão , Solubilidade
19.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1084-1095, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793902

RESUMO

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.


Assuntos
Proteínas de Transporte/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Proteínas Periplásmicas/química , Cinética , Modelos Moleculares , Conformação Proteica
20.
Proc Natl Acad Sci U S A ; 116(51): 25602-25613, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796585

RESUMO

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens.


Assuntos
Antígenos de Histocompatibilidade Classe I , Chaperonas Moleculares , Peptídeos , Dissulfetos/química , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Domínios Proteicos
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